A key issue in skin morphogenesis is how the topological arrangements of keratinocytes are organized to construct the complex three-dimensional appendage structures. Our long-term objective is to study the cellular and molecular basis of morphogenesis during the development and regeneration of skin appendages. We have been using chicken skin as the model for distinct forms, possibility for epithelial-mesenchymal recombination experiments and new technology for misexpressing genes in developing feathers. In the last funding period, we focused on early events of feather bud development. In this renewal, we will focus on late events in feather follicle morphogenesis, which have a complex, three level hierarchical branch structure and involve reciprocal interactions between feather stem cells and the dermal papilla. In Aim 1, we will study the axial determination of epithelial organs by analyzing how the bilaterally and radially symmetric feather follicle designs are built differently. We will test the tilting stem cell ring hypothesis that suggests the rachis and bilateral symmetry can be generated simply by tilting the ring of stem cells down toward the anterior end and creating a molecular gradient that breaks symmetry. Wnt and beta-catenin pathways are involved. In Aim 2, we will study how a cylindrical layer of feather filament keratinocytes can be "sculpted" into the elaborate barb/barbule branches with distinct structures and functions. We hypothesize that the geometric forms reflect the dazzling regulation of keratinocyte proliferation, apoptosis and differentiation functioning to its extreme (barbules are composed of a single row of keratinocytes), and that the length and characteristics of barbules mirror the antagonizing balance of molecular activities. BMP, Shh and Wnt pathways will be prioritized, among other pathways. In Aim 3, we will study the control of plasticity and specificity of different skin appendages. We propose that there is a gradual restriction of competence of the epidermis, coupled with a gradual increase of regional specificity conferred by the mesenchyme. FGF and T-box homeobox genes in feather / scale paradigm will be the model. These aims will be tested using the methodology mentioned above. Using this unique feather model, we hope to focus on the theme of molecular basis--> cell behavior--> biological forms. Learning about the regulation of keratinocytes at different levels may help us guide appendage stem cells to form the tissues and organs needed in tissue engineering.